A major challenge of contemporary neurobiology is our incomplete understanding of the mechanisms and plasticity involved in cell-to-cell communication. Determining how neurons communicate with other neurons within a network requires complete information about the neurotransmitters and neuropeptides present in and released from individual neurons. This proposal uses the well-known animal model Aplysia californica, with its well-defined neuronal networks, to characterize the suite of molecules used in individual neurons, as well as their release, in an activity dependent manner. By taking advantage of new analytical tools that allow single neurons and neuronal subcompartments to be assayed for their chemical constituents, combined with the data becoming available as part of the Aplysia genome and transcriptome projects, a nearly complete list of signaling molecules will be characterized from specific identified neurons involved in important physiological functions. For the classical transmitters, capillary electrophoresis with several selective detection schemes (ranging from radionuclide detection to native fluorescence) will be used. In particular, significant efforts will determine the roles of the unusual amino acids, d-glutamate and d-aspartate, in neurotransmission. While prior work has demonstrated that in specific neurons, these molecules are synthesized from their L-amino acid counterparts, transported to release zones and likely released, here the details of their functional roles in cell-to-cell signaling will be explored. In addition, the complete set of peptides (the peptidome) used in these neuronal networks will be characterized;the methods to be used include single cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, a variety of small volume electrospray mass spectrometric approaches, and several bioinformatics approaches. The outcome of this work will be a well-defined neurochemistry to complement the well-known physiology and behavior in the neuronal networks of Aplysia. By using the advances in separation science and mass spectrometry, significant gains can be made in our understanding of the synthesis, posttranslational processing, distribution, release and function of known and novel cell-to-cell signaling compounds. In leading to a description of the subcellular dynamics of neuronal signaling, which plays a crucial role in coordinating neuronal network activities, this work will contribute to furthering our basic understanding of the nervous system.